Process for preparing creatine amides

ABSTRACT

The invention relates to the field of pharmaceutical chemistry, specifically to processes for preparing biologically active substances (BAS), in particular creatine amides. What is proposed is a process for preparing creatine amides which comprises treating creatine with para-toluenesulfonic acid in an organic solvent with subsequent reaction of the resultant complex with compounds comprising a primary or secondary amino group in the presence of a condensing agent and a base which are introduced subsequently. The claimed process makes it possible to increase the yield of the end product by 2-5 times in comparison with analogs and also to extend the range of the compounds prepared.

FIELD OF INVENTION

The invention relates to pharmaceutical chemistry, more specifically to a methods of preparation of a new biologically active compounds, particularly to amides of creatine.

BACKGROUND OF THE INVENTION

Creatine is an endogenous nutrient occurring in a various mammalian tissues, for example, in the liver, kidneys, muscular tissue, brain tissue, blood and appears as well in the free form as in the form of creatine phosphate. Creatine is considered in a capacity of a remedy enhancing an energetic tissue metabolism, increasing an energetic reserve of ATP first of all in muscle and nerve cells.

Creatine phosphate (CrP) maintains ATP level upon increasing of energy consumption in a cell i.e. takes part in a physiological process of ADP phosphorylation with ATP formation. Among with glycogen CrP is one of main sourse of high-energy phosphates transformation cycle and so participates in an oxidative phosphorylation of glucose that provides an energy evolution essential for muscular tissue cells activity including skeletal muscles and cardiac muscle. Because of creatine phosphate is able to provide ATP biosynthesis an increasing of creatine amount in muscles increases a muscular supply of creatine phosphate, enhances an energetic tissue metabolism, enhances a muscles performance capability (tolerance), increases a muscles mass.

Creatine phosphate and creatine are also an allosteric regulators of cell processes (N. Brustovetsky et al., J. Neurochem. 2001. 76. 425-434). Thus administration of 20-30 g of creatine monohydrate per day for a few days leads to increasing of total creatine content in human skeletal muscles by more than 20%. Given properties attract the special attention in view of possibility to use creatine in a capacity of nutritional supplement for organism strengthening and increasing of performance capability. Thus administration of creatine monohydrate in an amount of 15 g per day for at least 2 days is used for an increasing the muscle performance ability (WO/94/002127).

Creatine and creatine phosphate find a wide application in medicine. Thus, creatine, creatine phosphate and cyclocreatine (U.S. Pat. No. 6,706,764) are recommended for a treatment of nervous system diseases such as diabetic and toxic neuropathies, Alzheimer's disease, Parkinson's disease, stroke, etc, metabolism disturbances such as hyperglycaemia and diabetes mellitus (U.S. Pat. No. 6,193,973). Per os administration of creatine is described for a treatment of cardiac insufficiency (WO/EP 97/06225), asthma (U.S. Pat. No. 6,093,746). Application of creatine phosphate was shown for a treatment of cardio-vascular diseases, prospectivity for a treatment of new-growth tissue (U.S. Pat. No. 5,219,846). In the mean time application of creatine and creatine phosphate are limited by bad solubility and stability in aqueous media at physiological pH values (RU 2 295 261).

Even more creatine is badly adsorbs from gastro-intestinal tract, extent of absorbtion consists a 1 to 14%. It makes necessary an administration of creatine in a high doses. For the purpose of creatine application was effective a composition manufactured at the present time is administered in amount of 20 g per day. In the mean time along with increasing of the cost of therapy administration of a high doses of creatine can lead to negative after-effects for an organism—nitrogen exchange deficit, gastro-intestinal diseases, diarrhea etc.

Therefore a preparation of creatine derivatives possessing the more stability or the more biological activity is of main interest that allows from the one hand to decrease a dose of administered compound and from another hand to find out a new field of application. Herein derivatives of creatine and variable organic acids attract the highest interest. Thus it is known that application of creatine pyruvates (U.S. Pat. No. 6,166,249; RU 2 114 823) for an enhancement of working efficiency, body weight decreasing, adaptation to oxygen unsufficiency associated with ischemia, in a capacity of nutritional supplement, for a protection of skin aging and sun light action (U.S. Pat. No. 7,186,754), for a treatment of female sexual disorders, in particular, dismenorrhea (U.S. Pat. No. 6,503,951).

Derivatives of creatine and malonic, maleic, fumaric, orotic acids and taurine (U.S. Pat. No. 6,838,562; U.S. Pat. No. 6,861,554; U.S. Pat. No. 6,166,249; U.S. Pat. No. 7,109,373) are indicated for nutritional care as a food supplement; creatine citrate (U.S. 2004/077719) is recommended in a capacity of nootropic agent.

One of the most perspective derivatives of creatine comprises amides of creatine of general formula NH═C(NH₂)—N(CH₃)—CH₂—CO—NH—R*X, wherein R is aminoacids residue or protected aminoacids residue; X—organic or mineral acid or water (RU 2 354 645) synthesized and studied by authors.

As it was determined in a process of experiments synthesized amides of creatine possessed an increased solubility and stability in aqueous solutions that allows to use it more wide in a capacity of creatine resource in an organism.

Method preparation of similar amides of creatine comprising of the most close to the claimed one upon achieved effect consist of interaction a deprotected and protected guanidilating agents with amides of sarcosine in polar organic solvents at a temperature not more than 50° C. Preparation of another derivatives of aminoacids is possible based on amides of sarcosine by using a standard chemical reactions described in literature (A. A. Gershkovich, V. K. Kibirev “Peptide Synthesis. Reagents and Methods. Kiev. “Naukova dumka”. 1987).

The disadvantage of given method are multistaging, associated with requirement amides of sarcosine preliminary preparation, insufficiently high yield of desired product which in particular for Creatinyl-Glycine Benzyl Ester Hydrochloride consists of about 5%.

SUMMARY OF THE INVENTION

A generation of more simple and effective technology of amides of creatine preparation with more high yield by conversion of creatine (Cr) to complex soluble in organic solvents as a result of its treatment by p-toluenesulfonic acid (p-TSA) upto complete dissolution of Cr followed by interaction of given complex with aminoacids derivatives containing a primary or secondary aminogroup in particular ester or amide derivatives of aliphatic or aromatic aminoacids in the presence of condensation agent and base consequently introduced is supposed.

MANUFACTURING PROCESS

Introduction of p-TSA carries out at least in an equimolar quantity relative to Creatine. Introduction of amino-component and condensation agent in solution carries out after a complete dissolution of creatine and p-TSA complex, and a base introduction—in 10-15 min since reaction starting and an acquisition of reaction equilibrium for equilibrium offset in a set way.

A problem in conversion of creatine in a solution and protection of guanidino-group by complex formation that complicates a conversion of creatine into creatinine as a result of intramolecular cyclization is dissolved simultaneously as a result of creatine treatment by p-TSA.

Creatine anhydrous or more low-cost Creatine hydrate were used in a capacity of Cr.

Carbodiimides particularly dicyclohexylcarbodiimide, N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide, diisopropylcarbodiimide or monoesters of chloroformic acid particularly ethylchloroformate, iso-butylchloroformate can be used in a capacity of condensation agent, and bases soluble in organic solvents, particularly, tertiary amines, f.e., N-morpholine, triethylamine, diisopropylamine etc., in a capacity of base binding of hydrochloric acid evolving. Dimethylformamide, dimethylacetamide, dimethylsulfoxide, N-methylpyrrolidone etc. can be used in a capacity of organic solvents providing a completed dissolution of creatine and p-TSA complex.

Using of monoesters of chloroformic acid, particularly iso-butylchloroformate or ethylchloroformate, and dicyclohexylcarbodiimide or diisopropylcarbodiimide in a capacity of carbodiimide are preferably in a capacity of condensation agent as a quantity of foreign impurities is decreased herein and a technology of a desired product isolation is simplified.

Reaction mixture obtained in a process purifies generally by using of ion-exchanged resin followed by recrystallization of desired product which yield achieves 20% with a purity of more than 90% by HPLC data. The basic impurity upon a synthesis is creatinine content of which in a desired product generally does not exceed of 1.5%.

Amides of creatine prepared in a process of claimed method can be modified further by using of organic synthesis standard technologies.

Control of reaction pathway and also purity evaluation of final products and intermediates are carried out by method of reverse phase HPLC on a chromatograph Alliance (Waters), column Zorbax ODS, 3.5 μm, 3×100 mm (Agilent Technologies). A mixture of buffer solution containing 0.01 M solution of sodium octansulfonate and 0.02 M of sodium dihydrogen phosphate with acetonitrile is used for eluation. Detection is carried out by method of UV-spectroscopy at wave-length 210 nm. Assay in amides of creatine is carried out by method of non-aqueous titration by HClO₄ at the presence of crystal violet as an indicator.

Molecular mass determination in amides of creatine is carried out by mass-spectrometry method at the time-of-flight mass-reflectrone MX-5303 with the ions source the “Electrospray” type (Division of Institute of Energy Problem in Chemical Physics, Russian Academy of Science).

DETAILED DESCRIPTION OF THE INVENTION Example 1 Preparation of Creatinyl-Glycine Iso-Propyl Ester Acetate

-   Suspension of 5.67 g (38 mM) of Creatine monohydrate in 40 mL of     N,N-Dimethylformamide was placed into a 250 mL 1-necked round bottom     flask charged with a pressure equalization arm dropping funnel     closed with calcium chloride tube then 7.23 g (38 mM) of     p-Toluenesulfonic acid monohydrate was added at stirring on a     magnetic stirrer. Creatine monohydrate is completely dissolved in 5     min. Then 6.71 g (40 mM) of Glycine iso-Propyl Ester Hydrochloride     was added and reaction mixture was cooled down to (−)10° C. at     ice-salted bath after its dissolution. Then 5.24 mL (38 mM) of     iso-Butylchloroformate was added and 8.9 mL (81 mM) of     N-Methylmorpholine was added via dropping funnel for 10 min.     Reaction mixture was stirred at ice bath for 1 hour then a     temperature was adjusted to room condition. N-Methylmorpholine     hydrochloride precipitate formed was filtered off in 10 hours, a     mother liquor was evaporated at 50° C. An oily residue was dissolved     in 160 mL of chloroform and kept at (−)10° C. for 10 hours. Solution     was filtered, extracted with H₂O (3×100 mL). A combined aqueous     phase containing a desired product was extracted with chloroform     twice, an aqueous solution was evaporated in vacuo up to volume of     50 mL. Resulted solution was flowed through a column 30×250 mm     filled with a Dowex 1×8 in acetate form in H₂O. Eluent—H₂O, elution     rate—2 mL/min. Column was eluted with H₂O monitoring a pH of eluate.     Fractions with a pH=7 were collected, analysed by Reverse Phase     HPLC, combined, an acetic acid was added and fractions were     evaporated. A residue was crystallized with 20 mL of acetonitrile at     (−)10° C. for 20 hours. A precipitate of Creatinyl-Glycine     iso-Propyl Ester Acetate was filtered off, washed with chilled     acetonitrile, diethyl ether and dried. A resulted product was     dissolved in 30 mL of ethanol, kept at (−)10° C. for 20 hours, a     solution was filtered, ethanol was removed in vacuo, a residue was     crystallized with diethyl ether. Yield of Creatinyl-Glycine     iso-Propyl Ester Acetate C₉H₁₈N₄O₃*C₂H₄O₂: 2.3 g (21%).     Mass-spectra, found: m/z 290.29. Calculated: M 290.32. Assay of     Creatinyl-Glycine iso-Propyl Ester Acetate by non-aqueous     titration—97.7% mass, creatinine impurity content—1.5% mass.

Example 2 Preparation of Creatinyl-Glycyl-Glycine Ethyl Ester Acetate

-   Suspension of 4.77 g (32 mM) of Creatine monohydrate in 40 mL of     N-Methylpyrrolidone was placed into a 250 mL 1-necked round bottom     flask charged with a pressure equalization arm dropping funnel     closed with calcium chloride tube, 5.02 g (35 mM) of     p-Toluenesulfonic acid anhydrous was added at stirring. Creatine     monohydrate is completely dissolved in 5 min. Then 4.33 g (22 mM) of     Glycyl-Glycine Ethyl Ester Hydrochloride was added and reaction     mixture was cooled down to (−)10° C. at ice-salted bath after its     dissolution. Then 4.51 mL (33 mM) of iso-Butylchloroformate was     added and 5.1 mL (54 mM) of N-Methylmorpholine was added via     dropping funnel for 10 min. Reaction mixture was stirred at ice bath     for 1 hour then a temperature was adjusted to room condition.     N-Methylmorpholine hydrochloride precipitate formed was filtered off     in 10 hours, a mother liquor was evaporated at 50° C. An oily     residue was dissolved in 100 mL of chloroform and kept at (−)10° C.     for 10 hours. Solution was filtered, extracted with H₂O (3×100 mL).     A combined aqueous phase containing a desired product was extracted     with chloroform twice and evaporated in vacuo up to volume of 50 mL.     Working up of desired product was carried out according to method     described in Example 1. Yield of C₁₀H₁₉N₅O₄*C2H₄O₂: 1.05 g (14.6%).     Mass-spectra, found: m/z 273.24. Calculated: M 273.29. Assay of     Creatinyl-Glycyl Glycine Ester Acetate by non-aqueous     titration—98.5% mass, creatinine impurity content—1.3% mass.

Example 3 Preparation of Creatinyl-Glycine Methyl Ester Hydrochloride

-   Suspension of 2.98 g (20 mM) of Creatine monohydrate in 20 mL of     Dimethylacetamide was placed into a 250 mL 1-necked round bottom     flask charged with a pressure equalization arm dropping funnel     closed with calcium chloride tube, then 3.99 g (21 mM) of     p-Toluenesulfonic acid monohydrate was added at stirring on a     magnetic stirrer. A precipitate is completely dissolved in 5 min.     Then 2.64 g (21 mM) of Glycine Methyl Ester Hydrochloride was added     and reaction mixture was cooled down to (−)10 ° C. at ice-salted     bath after its dissolution. Then 2.8 mL (20 mM) of     iso-Butylchloroformate was added and 4.4 mL (40 mM) of     N-Methylmorpholine was added via dropping funnel for 10 min.     Reaction mixture was stirred at ice bath for 1 hour then a     temperature was adjusted to room condition. N-Methylmorpholine     hydrochloride precipitate formed was filtered off in 7 hours, mother     liquor was treated with hexane to separate an oily precipitate. An     oily residue was dissolved in 80 mL of chloroform and kept at     (−)10° C. for 10 hours. Solution was filtered, extracted with H₂O     (3×100 mL). A combined aqueous phase containing a desired product     was extracted with chloroform (2×80 mL) and evaporated in vacuo up     to volume of 50 mL. Purification of a desired product was carried     out in an Example 1 condition with the exception of fractions with     pH=7, pH(H₂O)=5 were collected, analysed by Reverse Phase HPLC,     combined, hydrochloric acid was added and fractions were evaporated.     Yield of C₇H₁₄N₄O₃*HCl—1.52 g (30 %). Mass-spectra, found: m/z     238.65. Calculated: M 238.67. Assay of Creatinyl-Glycyl Glycine     Ester Hydrochloride by non-aqueous titration—98.3% mass, creatinine     impurity content—1.5% mass.

Example 4 Preparation of Creatinyl-Glycine Ethylamide Tartrate from Creatine Anhydrous

-   Suspension of 2.62 g (20 mM) of Creatine anhydrous in 20 mL of     N,N-Dimethylformamide was placed into a 250 mL 1-necked round bottom     flask charged with a pressure equalization arm dropping funnel     closed with calcium chloride tube, then 3.83 g (20.11 mM) of     p-Toluenesulfonic acid monohydrate was added at stirring on a     magnetic stirrer. A precipitate is completely dissolved in 10 min.     Then 4.77 g (22 mM) of Glycine Ethylamide trifluoroacetate was added     and reaction mixture was cooled down to (−)10° C. at ice-salted bath     after its dissolution. Then 2.8 mL (20 mM) of iso-Butylchloroformate     was added and 4.65 mL (43 mM) of N-Methylmorpholine was added via     dropping funnel for 10 min. Reaction mixture was stirred at ice bath     for 1 hour then a temperature was adjusted to room condition. A     precipitate formed was filtered off in 20 hours, mother liquor was     evaporated in vacuo at 50° C. An oily residue was dissolved in 80 mL     of chloroform and kept at (−)10° C. for 20 hours. Solution was     filtered, extracted with H₂O (3×100 mL), a combined aqueous phase     containing a desired product was extracted with chloroform (2×80 mL)     and evaporated in vacuo up to volume of 50 mL. Resulted product was     isolated by method performed in Example 1, with the exception of     column with Dowex 1×8 in tartaric form was used. Fractions with pH=7     were collected, analysed by Reverse Phase HPLC, combined,     evaporated. Yield of [C₈H₁₇N₅O₂]₂*C₄H₆O₆—3.7 g (32%). Mass-spectra,     found: m/z 583.62. Calculated: M 583.61. Assay of Creatinyl-Glycine     Ethylamide Tartrate by non-aqueous titration —98.9% mass, creatinine     impurity content—0.7% mass.

Example 5 Preparation of N^(ε)-Creatinyl-Lysine Ethyl Ester Diacetate

-   Suspension of 3.0 g (20.5 mM) of Creatine monohydrate in 40 mL of     N,N-Dimethylformamide was placed into a 250 mL 1-necked round bottom     flask charged with a pressure equalization arm dropping funnel     closed with calcium chloride tube and 3.81 g (20.5 mM) of     p-Toluenesulfonic acid monohydrate was added at stirring. Then 7.0 g     (20.5 mM) of α-Carbobenzoxy-L-Lysine Ethyl Ester hydrochloride was     added and reaction mixture was cooled down to (−)10° C. at     ice-salted bath after its dissolution. Then 2.8 mL (20.5 mM) of     iso-Butylchloroformate was added and 4.4 mL (20.5 mM) of     N-Methylmorpholine was added via dropping funnel for 10 min.     Reaction mixture was stirred at ice bath for 1 hour then a     temperature was adjusted to room condition. Suspension was filtered,     mother liquor was evaporated in vacuo at 50° C. An oily residue was     dissolved in 150 mL of n-butanol, washed with NaHCO₃ 5% four times     then with H₂O twice. Butanol was evaporated, 30 mL of H₂O was added     to a residue and resulted suspension was applied onto column with     sorbent YMC*Gel ODS 22.5×150 equilibrated with 0.2% aqueous solution     of acetic acid; column was eluted with 0.2% aqueous solution of     acetic acid then eluted in a gradient rate upto 10% of iso-propyl     alcohol in 0.2% aqueous solution of acetic acid. Fractions were     analysed by qualitative reaction and HPLC, combined and evaporated.     A residue was crystallized with 5 mL of acetonitrile, filtered off     and dried in vacuo. Yield of     C₂₀H₃₁N₅O₅*C₂H₄O₂-N^(α−)Carbobenzoxy-N^(ε)-Creatinyl-Lysine Ethyl     Ester Acetate 1.5 g (18%). Mass-spectra, found: m/z 481.77.     Calculated: M 481.77. Assay of     N^(α−)Carbobenzoxy-N^(ε)-Creatinyl-Lysine Ethyl Ester Acetate by     non-aqueous titration—98.9% mass, creatinine impurity content—1.0%     mass. -   1.0 g of N^(α−)Carbobenzoxy-N^(ε)-Creatinyl-Lysine Ethyl Ester     Acetate prepared was dissolved in 10 mL of methanol and hydrogenated     over Pd black for 3 hours. The completeness of carbobenzoxy-group     leaving was controlled by TLC in the system acetonitrile-H₂O-acetic     acid (6:1:1). Catalyst was filtered off, 0.5 mL of acetic acid was     added to a mother liquor and evaporated. Product was filtered off,     washed with chilled acetonitrile, diethyl ether and dried in vacuo.     Yield of C₁₂H₂₅N₅O₃*2C₂H₄O₂-N^(ε)-Creatinyl-Lysine Ethyl Ester     diacetate 0.2 g (42%). Mass-spectra, found: m/z 407.34. Calculated:     M 407.46. Assay of N^(ε)-Creatinyl-Lysine Ethyl Ester diacetate by     non-aqueous titration—99.2% mass, creatinine impurity content—0.5%     mass.

Example 6 Preparation of Creatinyl-Phenylalanine Ethylamide Hydrochloride from Creatine Anhydrous

-   Suspension of 2.62 g (20 mM) of Creatine monohydrate in 20 mL of     Dimethylsulfoxide was placed into a 250 mL 1-necked round bottom     flask charged with a pressure equalization arm dropping funnel     closed with calcium chloride tube and 3.83 g (20.11 mM) of     p-Toluenesulfonic acid monohydrate was added at stirring on a     magnetic stirrer. A precipitate is completely dissolved in 10 min.     Then 6.71 g (20 mM) of Phenylalanine ethylamide trifluoroacetate was     added reaction mixture was cooled down to (−)10° C. at ice-salted     bath after its dissolution. Then 2.8 mL (20 mM) of     iso-Butylchloroformate was added and 4.65 mL (43 mL) of morpholine.     Reaction mixture was stirred at ice bath for 1 hour then a     temperature was adjusted to room condition. Precipitate formed was     filtered off, a mother liquor was evaporated at 50° C. An oily     residue was dissolved in 80 mL of chloroform and kept at (−)10° C.     for 20 hours. Solution was filtered and extracted with H₂O (3×100     mL), a combined aqueous phase containing a desired product was     extracted with chloroform (2×80 mL) and evaporated in vacuo up to     volume of 50 mL. Resulted product was worked up by method performed     in Example 1, with the exception of fractions with pH=7 were     collected, analysed by Reverse Phase HPLC, combined, 1 M solution of     hydrochloric acid was added and fractions were evaporated. Yield of     C₁₅H₂₃N₅O₂*HCl—Creatinyl-Phenylalanine Ethylamide Hydrochloride 3.7     g (32%). Mass-spectra, found: m/z 341.87. Calculated: M 341.84.     Assay of Creatinyl-Phenylalanine Ethylamide Hydrochloride by     non-aqueous titration—99.3% mass, creatinine impurity content—0.5%     mass.

Example 7 Preparation of Creatinyl-Glycyl-Alanine Ethyl Ester Hemisuccinate

-   Suspension of 2.0 g (13.4 mM) of Creatine monohydrate in 10 mL of     Dimethylformamide was placed into a 100 mL 1-necked round bottom     flask charged with a pressure equalization arm dropping funnel     closed with calcium chloride tube and 2.54 g (13.4 mM) of     p-Toluenesulfonic acid monohydrate was added at stirring on a     magnetic stirrer and then 1.41 g (6.7 mM) of Glycyl-Alanine Ethyl     Ester Hydrochloride, 1.38 g (6.7 mM) of dicyclohexylcarbodiimide in     2 mL of Dimethylformamide. Then 1.14 mL (6.7 mM) of     diisopropylethylamide was added via dropping funnel in 10 min.     Reaction mixture was kept for 20 hours at room temperature, a solid     of dicyclohexylcarbodiimide hydrochloride and dicyclohexylurea     hydrochloride was filtered off, a mother liquor was evaporated at     50° C. An oily residue was dissolved in -   100 mL of chloroform and extracted with H₂O (3×100 mL). A combined     aqueous phase containing a desired product was extracted with     chloroform twice and evaporated in vacuo up to volume of 20 mL. A     resulted solution was eluted with H₂O through a column 30×150 mm     with Dowex 2×8 in succinic form. Elution rate was 2 mL/min. A column     was eluted with H₂O monitoring a pH value of eluent. Fraction with     pH 6÷7 were collected, combined and evaporated. A residue was     crystallized with 10 mL of acetonitrile at (−)10° C. for 5 hours. A     solid of Creatinyl-Glycyl-Alanine Ethyl Ester Hemisuccinate was     filtered off, washed with chilled acetonitrile, diethyl ether and     dried. A yield of crude product was 1.8 g. A product was dissolved     in 10 mL of ethanol, kept at (−)10° C. for 10 hours, a solution was     filtered. A mother liquor was evaporated, a residue was dissolved in     5 mL of H₂O and applied onto column with sorbent YMC*Gel ODS     22.5×150 equilibrated with 0.05% aqueous solution of succinic acid.     A column was eluted with 0.2% succinic acid, fractions were     monitored by HPLC. Fractions containing a desired product with a     purity not less then 95% were evaporated. A residue was dried by     distillation with iso-propanol and crystallized with diethyl ether.     Yield of C₁₁H₂₁N₅O₄*0.5 C₄H₆O₄-Creatinyl-Glycyl-Alanine Ethyl Ester     Hemisuccinate—0.56 g (24%). Mass-spectra, found: m/z 346.33.     Calculated: M 346.36. Assay of Creatinyl-Glycyl-Alanine Ethyl Ester     Hemisuccinate by non-aqueous titration—99.4% mass, creatinine     impurity content by HPLC—0.8% mass.

Example 8 Preparation of Creatinyl-γ-Aminobutyric Acid Ethyl Ester Acetate

-   Suspension of 4.20 g (32 mM) of Creatine in 40 mL of     Dimethylformamide was placed into a 250 mL 1-necked round bottom     flask charged with a pressure equalization arm dropping funnel     closed with calcium chloride tube and 5.02 g (35 mM) of     p-Toluenesulfonic acid monohydrate was added at stirring, 5.36 g (32     mM) of γ-aminobutyric acid ethyl ester hydrochloride, and reaction     mixture was cooled down to (−)10° C. at ice-salted bath after its     dissolution. Then 3.2 mL (33 mM) of ethylchloroformate was added,     5.1 mL (54 mM) of N-Methylmorpholine was added via dropping funnel     for 10 min. Reaction mixture was stirred at ice bath for 1 hour then     a temperature was adjusted to room condition. A solid of     N-Methylmorpholine hydrochloride formed was filtered off, a mother     liquor was evaporated at 50° C. A solution was recrystallized with     iso-propanol, purified by ion-exchanged chromatography on a column     with Sephadex SE-C25 in pyridine-acetate buffer solution. Fractions     containing of a desired product were combined, evaporated. Yield of     C₁₀H₂₀N₄O₃*C₂H₄O2-Creatinyl-γ-aminobutyric acid ethyl ester     acetate—1.95 g (25.0%). Mass-spectra, found: m/z 304.34. Calculated:     M 304.35. Assay of Creatinyl-γ-aminobutyric acid ethyl ester acetate     by non-aqueous titration—98.7% mass, creatinine impurity content by     HPLC—1.2% mass.

Example 9 Preparation of Creatinyl-Alanine Ethyl Ester Acetate

-   Suspension of 2.0 g (13.4 mM) of Creatine monohydrate in 10 mL of     Dimethylformamide was placed into a 100 mL 1-necked round bottom     flask charged with a pressure equalization arm dropping funnel     closed with calcium chloride tube and 2.54 g (13.4 mM) of     p-Toluenesulfonic acid was added at a stirring on a magnetic stirrer     and then 1.01 g (6.7 mM) of Alanine Ethyl Ester hydrochloride, 0.85     g (6.7 mM) of diisopropylcarbodiimide in 2 mL of Dimethylformamide.     Then 0.94 mL (6.7 mM) of triethylamine was added via dropping funnel     for 10 min. Reaction mixture was kept for 20 hours at a room     temperature, a solid of diisopropylethylamine and diisopropylurea     hydrochlorides was filtered off, a mother liquor was evaporated at     50° C. An oily residue was dissolved in 100 mL of chloroform and     extracted with H₂O (3×100 mL). A combined aqueous phase containing a     desired product was extracted with chloroform twice and evaporated     at rotor evaporator to volume of 20 mL. A resulted solution was     eluted with H₂O through a column 30×150 mm with Dowex 2×8 in acetate     form. Elution rate was 2 mL/min. A column was eluted with H₂O     monitoring a pH of eluate. Fractions with pH 6÷7 were collected,     combined, evaporated. A residue was crystallized with 10 mL of     acetonitrile at (−)10° C. for 5 hours. A solid of Creatinyl-Alanine     Ethyl Ester acetate was filtered off, washed with chilled     acetonitrile, diethyl ether and dried. Yield of     C₉H₁₈N₄O₃*C₂H₄O₄-Creatinyl-Alanine Ethyl Ester acetate—0.56 g (24%).     Mass-spectra, found: m/z 290.35. Calculated: M 290.31. Assay of     Creatinyl-glycylalanine ethyl ester acetate by non-aqueous     titration—99.1%, creatinine impurity content by HPLC—0.9% mass.

Example 10 Preparation of Creatinyl-Phenylalanine Ethyl Ester Acetate

-   Suspension of 11.3 g (86 mM) of Creatine in 80 mL of     Dimethylformamide was placed into a 250 mL 1-necked round bottom     flask charged with a pressure equalization arm dropping funnel     closed with calcium chloride tube and 16 g (86 mM) of     p-Toluenesulfonic acid monohydrate was added at a stirring on a     magnetic stirrer. A precipitate is completely dissolved in 5 min.     Then 11.5 g (50 mM) of Phenylalanine Ethyl Ester hydrochloride and     7.75 mL (45 mM) of diisopropylcarbodiimide were added. Then 8.6 mL     (50 mM) of diisopropylethylamine was added via dropping funnel for     10 min. Reaction mixture was kept for 20 hours at a room temperature     then 10 mL of H₂O was added, a solid of diisopropylurea formed was     filtered off, a resulted solution was analysed by HPLC. Reaction has     been completed at 50%. -   A resulted product was isolated by method presented at the Example 1     with exception of that fractions with pH=7, pH(H₂O)=5 were     collected, analysed by Reversed Phase HPLC, combined, 1 M solution     of acetic acid was added and evaporated. Yield of     C₁₅H₂₂N₄O₃*C₂H₄O-Creatinyl-Phenylalanine Ethyl Ester Acetate—7.9 g     (25%). Mass-spectra, found: m/z 366.37. Calculated: M 366.41. Assay     of Creatinyl-Phenylalanine Ethyl Ester acetate by non-aqueous     titration—99.4%, creatinine impurity content by HPLC—0.7% mass.

Study of Amides of Creatine Neuroprotective Activity

Research here and after was carried out by using the following amides of creatine prepared by claimed method:

-   1. Creatinyl-Glycine iso-Propyl Ester Acetate -   2. Creatinyl-Glycyl-Glycine Ethyl Ester Acetate -   3. Creatinyl-Glycine Ethylamide Tartrate -   4. N^(ε)-Creatinyl-Lysine Ethyl Ester Diacetate -   5. Creatinyl-Phenylalanine Ethylamide Hydrochloride -   6. Creatinyl -γ-Aminobutyric acid Ethyl Ester acetate.

Research in neuroprotective action of amides of creatine was carried out in rat model of focal ischemia (male, Wistar, 12÷14 weeks, 220÷240 g). Anesthesia was performed by sodium thiopental 60 mg/kg. A solution of amides of creatine in saline was administered intravenous to the first group of animals 45 min before ischemia, saline was administered to a control group, a solution of amides of creatine in saline was administered intragastric per os by an enteral tube a day before ischemia to the second group of animals 3 times per day, saline was administered to a control group.

Endovascular occlusion of middle cerebral artery was carried out by Koizumi J. et al (1986) method in modification of Longa E. Z. et al (1989) and Belayev L. et al (1999). Standard time of ischemia was consisted of 39 min, of reperfusion—48 hour. Animals were sacrificed, brain slices with thickness of 2 mm were produced for assess of brain damage volume. Damage area was determined by staining with phenyltetrazolium chloride followed by data acquisition and analysis of stained slice digital images. Brain damage coefficient—relation of damage area to slice surface—was calculated (Table 1).

TABLE 1 Effect of amides of creatine on a brain damage in male rat Wistar associated with experimental ischemia/reperfusion (p < 0.05). Method of administration, dose Agent (n = 5) Brain damage coefficient, % Agent 1 i./v.; 150 mg/kg 11.2 ± 2.3 Agent 2 i./v.; 200 mg/kg 12.1 ± 2.0 Agent 3 i./v.; 150 mg/kg 12.0 ± 1.7 Agent 4 i./v.; 100 mg/kg 13.3 ± 3.2 Agent 5 i./v.; 50 mg/kg 14.0 ± 1.1 Agent 6 i./v.; 45 mg/kg 13.1 ± 1.0 Saline i./v. 19.4 ± 5.3 Agent 1 per os; 3 × 150 mg/kg 14.2 ± 3.0 Agent 3 per os; 3 × 150 mg/kg 13.9 ± 3.3 Agent 4 per os; 3 × 100 mg/kg 15.2 ± 2.9 Saline per os 21.4 ± 4.7

Study of Amides of Creatine Effect on a Retention of Cognitive Function Associated with Cerebral Ischemia

-   Cerebral ischemia was induced by the method mentioned above. A     solution of amides of creatine or saline was administered     intravenous. Garcia et al (1955) score was used for neurological     deficit assess associated with focal ischemic and reperfusion     cerebral damage. Testing in animals was carried out before ischemia,     on a second and third days after ischemia-reperfusion at a fixed     time for exclusion of behavioral changes by means of circadian     rhythm. Spontaneous activity, symmetrical limb movement, climbing     onto vertical reticulated wall, body proprioreception, touching to     vibriss were assessed. Maximum point by assigned score was 18 (lack     of neurological deficit), minimum—3 (severe neurological deficit).     Results were listed in a Table 2.

TABLE 2 Neurological condition in rat by Garcia score on 2^(nd) and 3^(rd) days after ischemia (p < 0.05). Neurological condition by Garcia score 1 day before 2^(nd) day after 3^(rd) day afte Agent, dose ischemia ischemia r ischemia Agent 1; 150 mg/kg 18 ± 0 10 ± 2 15 ± 2 Agent 3; 150 mg/kg 18 ± 0 11 ± 2 16 ± 1 Agent 4; 100 mg/kg 18 ± 0 14 ± 1 17 ± 2 Agent 5; 50 mg/kg 18 ± 0 11 ± 1 14 ± 1 Agent 6; 45 mg/kg 18 ± 0 14 ± 0 16.0 ± 1.1 Saline 18 ± 0  6 ± 1  9 ± 2

Study of Amides of Creatine Stability in Artificial Gastric Secretion and Human Plasma.

-   10 mg of amides of creatine was dissolved in 20 mL of artificial     gastric secretion for study of amides of creatine stability,     solution aliquot was place into vial for chromatograph. Vial with     solution was place into chromatograph autosampler at 37° C. Initial     concentration of amides of creatine and its relative change in     gastric secretion were determined by HPLC at indicated temperature     (Table 3).

TABLE 3 Stability of creatine analoques in gastric secretion at a temperatute of 37° C. Stability, % Agent 0 hour 1 hour 3 hours 5 hours Agent 1 100 100 99 97 Agent 2 100 100 98 98 Agent 3 100 100 97 98 Agent 4 100 100 98 97 Agent 5 100 100 98 99 Agent 6 100 100 99 99

-   Stability of amides of creatine in human plasma was assessed by     relative change in concentration of amides of creatine in plasma. 1     mL of plasma was mixed with 0.2 mL of amides of creatine aqueous     solution with concentration 5 mg/mL, a mixture was kept at a     temperature of 37° C., aliquot of volume 0.2 mL was drawn in fixed     time ranges, mixed with 0.02 mL of trifluoroacetic acid 10%     solution, a precipitate was centrifuged at 3 00 g for 20 min. A     concentration of amides of creatine in supernatant was determined by     HPLC method (Table 4).

TABLE 4 Stability of creatine analoques in human plasma at a temperatute of 37° C. Stability, % Agent 0 hour 0.25 hour 0.5 hours 1 hour Agent 1 100 100 99 91 Agent 2 100 100 97 90 Agent 3 100 100 95 90 Agent 4 100 100 100 89 Agent 5 100 100 97 87

-   Represented data has shown that amides of creatine can be prepared     by using a claimed method by more simple technology, from more cheap     raw material and with more high yield. Prepared amides of creatine     is of interest for an application in medicine as it possess a     neuroprotective action. 

1. A method of preparation of amides of creatine, comprising of Creatine treatment by p-Toluenesulfonic acid in organic solvent followed by interaction of prepared complex with aminoacids derivatives containing a primary or a secondary aminogroup in the presence of condensation agent such as carbodiimide or monoesters of chloroformic acid and base such as tertial amines.
 2. A method of claim 1, wherein a treatment by p-Toluenesulfonic acid is carried out at least in an equimolar quantity relative to Creatine.
 3. A method of claim 1, wherein Creatine is Creatine anhydrous.
 4. A method of claim 1, wherein Creatine is Creatine monohydrate.
 5. A method of claim 1, wherein derivatives of aminoacids are ester or amide derivatives of aliphatic or aromatic aminoacids.
 6. A method of claim 1, wherein dicyclohexylcarbodiimide is used in a capacity of condensation agent.
 7. A method of claim 1, wherein diisopropylcarbodiimide is used in a capacity of condensation agent.
 8. A method of claim 1, wherein iso-Butylchloroformate is used in a capacity of condensation agent.
 9. A method of claim 1, wherein Ethylchloroformate is used in a capacity of condensation agent.
 10. A method of claim 1, wherein base soluble in organic solvents in a quantity providing a binding of hydrochloric acid evolving is used in a capacity of base.
 11. A method of claim 1, wherein N-methylmorpholine is used in a capacity of base.
 12. A method of claim 1, wherein triethylamine is used in a capacity of base.
 13. A method of claim 1, wherein diisopropylamine is used in a capacity of base.
 14. A method of claim 1, wherein Dimethylacetamide, N-Methylpyrrolidone, Dimethylsulfoxide, N,N-Dimethylformamide are used in a capacity of an organic solvent. 